title: PPP1CC2 can form a kinase/phosphatase complex with the testis-specific proteins TSSK1 and TSKS in the mouse testis.
abstract: The mouse protein phosphatase gene Ppp1cc is essential for male fertility, with mutants displaying a failure in spermatogenesis including a widespread loss of post-meiotic germ cells and abnormalities in the mitochondrial sheath. This phenotype is hypothesized to be responsible for the loss of the testis-specific isoform PPP1CC2. To identify PPP1CC2-interacting proteins with a function in spermatogenesis, we carried out GST pull-down assays in mouse testis lysates. Amongst the identified candidate interactors was the testis-specific protein kinase TSSK1, which is also essential for male fertility. Subsequent interaction experiments confirmed the capability of PPP1CC2 to form a complex with TSSK1 mediated by the direct interaction of each with the kinase substrate protein TSKS. Interaction between PPP1CC2 and TSKS is mediated through an RVxF docking motif on the TSKS surface. Phosphoproteomic analysis of the mouse testis identified a novel serine phosphorylation site within the TSKS RVxF motif that appears to negatively regulate binding to PPP1CC2. Immunohistochemical analysis of TSSK1 and TSKS in the Ppp1cc mutant testis showed reduced accumulation to distinct cytoplasmic foci and other abnormalities in their distribution consistent with the loss of germ cells and seminiferous tubule disorganization observed in the Ppp1cc mutant phenotype. A comparison of Ppp1cc and Tssk1/2 knockout phenotypes via electron microscopy revealed similar abnormalities in the morphology of the mitochondrial sheath. These data demonstrate a novel kinase/phosphatase complex in the testis that could play a critical role in the completion of spermatogenesis.
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, Ontario, Canada M5S 3G5.
authors.forename: Graham
authors.initials: G
authors.lastname: MacLeod
authors.forename: Peng
authors.initials: P
authors.lastname: Shang
authors.forename: Gregory T
authors.initials: GT
authors.lastname: Booth
authors.forename: Lucas A
authors.initials: LA
authors.lastname: Mastropaolo
authors.forename: Niloufar
authors.initials: N
authors.lastname: Manafpoursakha
authors.forename: A Wayne
authors.initials: AW
authors.lastname: Vogl
authors.forename: Susannah
authors.initials: S
authors.lastname: Varmuza
mesh_terms: D000818:Animals; D003598:Cytoskeletal Proteins; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D054645:Protein Phosphatase 1 / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D013091:Spermatogenesis / Q000502:physiology; D013737:Testis / Q000378:metabolism*
chemical_list: D003598:Cytoskeletal Proteins; D010750:Phosphoproteins; C530613:TSKS protein, mouse; C475219:Tsk1 protein, mouse; D017346:Protein Serine-Threonine Kinases; C517103:Ppp1cc protein, mouse; D054645:Protein Phosphatase 1